Spheroids have emerged as powerful tools in drug screening by providing a physiologically relevant environment that more closely mimics in vivo conditions compared to 2D cultures. Despite these advantages, experimental outcomes using spheroids are often influenced by considerable variability and bias. This review explores three key factors that affect the evaluation of drug efficacy in spheroids, including fabrication method, spheroid size, and cell viability. First, it compares five commonly used spheroid fabrication platforms, outlining their principles, advantages, and limitations. Second, it examines biological contributors to spheroid size variation, such as cell proliferation, apoptosis, protein expression, and cellular differentiation, and how they affect drug response. Third, it discusses how features such as hypoxic core development and anoikis sensitivity affect cell viability and potentially distort toxicity assessments. This review presents a conceptual framework for minimizing bias and improving reproducibility in spheroid-based drug screening.